LAB 2 WRITE-UP

Thermal Cycler Engineering

Our re-design is based upon the Open PCR system originally designed by Josh Perfetto and Tito Jankowski.

System Design

Key Features

Instructions

Protocols

Materials

Supplied in the Kit

Amount

Open PCR Machine

1

10 μM Forward Primer

16.0 μL

10 μM Reverse Primer

16.0 μL

GoTaq Master Mix

800.0 μL

dH2O

764.8 μL

Eppendorf Tubes

16

Fluorimeter

1

Teflon Glass Slides

16

Allen Wrench

1

Operations Manual

1

Supplied by User

Amount

Screwdriver

1

Template DNA (20ng)

3.2 μL

Micro-Pipetter

16

Camera Phone

1

PCR Protocol

1. Gather materials and assemble as shown in apparatus/manual
2. Label provided tubes with a number to indicate its sample.
4. With the micropipette, place the forward/reverse primer into the indicated tube.
5. Use a clean micropipette to place the master mix into the DNA/primer mixture in the tubes.
6. Transfer the 16 Eppendorf tubes containing DNA and Mix into the Open PCR Machine.
7. Open PCR program and input the number of cycles: 30.
8. Set the temperature and times into the program.
- Denaturing temperature to 95°C. Time to 30 seconds.
-Annealing temperature to 57°C. Time to 30 seconds.
-Extending temperature to 72°C. Time to 30 seconds.
9. Close lid to commence process.
10. Start program.
11. When PCR is complete, ensue to the DNA Measurement Protocol shown below.

DNA Measurement Protocol

1. Create 8 DNA template samples that will be the focus of the investigation and place them in the PCR Machine allowing them to complete the process and replicate.

2. Transfer each sample independently into its designated Eppendorf Tube with the 400mL buffer completely. Ensure that a single pipette is used per sample.

3. After setting up the fluorimeter, place 2 drops of SYBR Green onto the teflon slide followed by 2 drops of the sample you wish to use. (Again use the same pipette used to transfer the sample)

4. Align up the drop so the light is passing through it.

5. Take pictures of the drop using a camera or smartphone, these will later be uploaded to ImageJ for analysis.

6. Each slide is capable of handling 5 individual samples so simply place the drops in an empty space and repeat the process.

7. Also run drops from the scintillation vial as blanks using the same process.

8. Upload the pictures taken into Image J.

9. To analyze the images subtract the INTDEN measurement form the background from the INTDEN measurement form the drop and repeat for all trials.

Sickle cell anemia is an inherited blood disorder characterized primarily by chronic anemia and periodic episodes of pain. The underlying problem involves hemoglobin, a component of red blood cells. Hemoglobin molecules in each red blood cell carry oxygen from the lungs to body organs and tissues and bring carbon dioxide back to the lungs.

In sickle cell anemia, the hemoglobin is defective. After hemoglobin molecules give up their oxygen, some may cluster together and form long, rod-like structures. These structures cause red blood cells to become stiff and assume a sickle shape.

Unlike normal red cells, which are usually smooth and donut-shaped, sickled red cells cannot squeeze through small blood vessels. Instead, they stack up and cause blockages that deprive organs and tissues of oxygen-carrying blood. This process produces periodic episodes of pain and ultimately can damage tissues and vital organs and lead to other serious medical problems. Normal red blood cells live about 120 days in the bloodstream, but sickled red cells die after about 10 to 20 days. Because they cannot be replaced fast enough, the blood is chronically short of red blood cells, a condition called anemia.

Inheritance

Sickle cell anemia is an autosomal recessive genetic disorder caused by a defect in the HBB gene, which codes for hemoglobin. The presence of two defective genes (SS) is needed for sickle cell anemia. If each parent carries one sickle hemoglobin gene (S) and one normal gene (A), each child has a 25% chance of inheriting two defective genes and having sickle cell anemia; a 25% chance of inheriting two normal genes and not having the disease; and a 50% chance of being an unaffected carrier like the parents.